Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system.

The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5-6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications.

The cytoplasmic domains of complement regulatory protein CD46 interact with multiple kinases in macrophages.

Membrane cofactor protein (CD46), which normally protects autologous cells from complement lysis, is the human cell receptor for measles virus (MV). Interaction between MV and CD46 on monocytes can lead to suppression of monocyte activation. We have investigated the interaction between the cytoplasmic sequences of CD46 and kinases in a mouse macrophage cell line. Glutathione-S-transferase (GST) fusion proteins bearing the Cyt1 or Cyt2 alternative cytoplasmic domain of CD46 associate with macrophage kinase activity, which phosphorylates multiple proteins co-purified with the GST fusion proteins. Association with the macrophage kinase activity correlates with tyrosine phosphorylation of the CD46 cytoplasmic domains. Removing the CD46 sequences or introducing a frame-shift mutation abrogates the association with macrophage kinase activity. Renaturation studies reveal multiple kinases with apparent molecular mass of 82, 79, 58, and 50/49 kDa, which associate specifically with both CD46 cytoplasmic domains. Alanine substitutions at a juxtamembrane Tyr-X-X-Leu motif in the Cyt1 domain completely abrogate the association with macrophage kinases and tyrosine phosphorylation of Cyt1; but similar substitutions in the Cyt2 domain only partially reduce the association with kinases and tyrosine phosphorylation of Cyt2. These results reveal a specific interaction between complement regulatory protein CD46 and macrophage kinases. These findings may provide an important clue for understanding immune modulation by MV.

Genomic progression in mouse models for liver tumors.

The principal cause of human liver cancer is infection with hepatitis viruses B and C, but tumor progression is fueled by ensuing perturbations that confer gain of function on proto-oncogenes or loss of function on tumor suppressor genes. Frequent among these perturbations is overexpression of the proto-oncogene MET. We have modeled the pathogenesis of liver tumors by expressing conditional transgenes of MET in the hepatocytes of inbred mice. The response to the MET transgene varied with both the magnitude and timing of its expression but included hyperplasia of hepatic progenitor cells, as well as benign and malignant tumors that display both phenotypic and genotypic resemblances to human counterparts. The results reveal MET to be a crucial switch in the development of the liver; dramatize how different cellular compartments within a developmental lineage can give rise to distinctive tumor stem cells; delineate rules of tumor progression; provide evidence that the experimental tumors in mice are authentic models for human tumors; and support a role for MET in the genesis of human liver tumors. The models should be useful in elucidating the mechanisms of tumorigenesis and in the preclinical testing of new therapeutics.

Identification of a cytoplasmic Tyr-X-X-Leu motif essential for down regulation of the human cell receptor CD46 in persistent measles virus infection.

To investigate the sequence requirements for measles virus (MV)-induced receptor down regulation, we transfected the human CD46 gene into simian cells persistently infected by the Biken strain of MV. Surface expression of CD46 is drastically reduced in these cells. Deletion analysis has shown that the juxtamembrane region of the CD46 cytoplasmic domain is essential for down regulation. Deleting a Tyr-Arg-Tyr-Leu sequence in this region or changing these residues to Ala prevents CD46 down regulation from the infected cell surface. Alanine-scanning mutagenesis has identified two amino acid residues, Tyr and Leu, forming a Tyr-X-X-Leu motif critical for CD46 down regulation. Mutations that prevent CD46 down regulation enhance syncytium formation. These results indicate that CD46 down regulation limits the cytopathic effects in a persistent MV infection and that CD46 down regulation requires a cytoplasmic Tyr-X-X-Leu sequence which resembles known motifs for membrane protein trafficking and receptor signalling.

Expression of human complement regulatory protein CD46 restricts measles virus replication in mouse macrophages.

Measles virus (MV) can infect mouse macrophages to cause a prolonged non-cytopathic infection that produces low levels of infectious virus for days. We have generated RAW264.7 mouse macrophages expressing human CD46, a cell surface complement regulatory protein that serves as a receptor for laboratory-adapted strains of MV. Laboratory-adapted MV strains efficiently enter the CD46-positive mouse macrophages to cause a cytopathic infection with extensive multinucleated cells and pseudopodia-like extensions. However, MV infection of mouse macrophages through CD46 is self-limiting. Both viral protein synthesis and infectious virus production are abruptly terminated after the second day of infection. This novel virus-cell interaction is seen only in mouse macrophages but not in mouse or hamster fibroblasts expressing human CD46. The possible role of CD46 in macrophage antiviral response restricting MV replication is discussed.

Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver.

The short duration of transgene expression remains a major obstacle for the implementation of nonviral DNA vectors in clinical gene therapy trials. Here, we demonstrate stable, long-term transgene expression in vivo by transfecting a linear DNA expression cassette (LDNA) into mouse liver. Interestingly, despite similar quantities and cellular distribution of injected DNAs in their livers, mice receiving LDNA encoding human alpha1-antitrypsin (hAAT) expressed approximately 10- to 100-fold more serum hAAT than mice injected with closed circular (cc) DNA for a period of 9 months (length of study). Furthermore, when a linear human factor IX expression cassette was delivered to factor IX-deficient mice, sustained serum concentrations of more than 4 microg/ml (80% of normal) of the human clotting factor and correction of the bleeding diathesis were obtained. Southern blot analyses indicate that, unlike ccDNA, LDNA rapidly formed large, unintegrated concatemers in vivo, suggesting that transgene persistence from plasmid-based vectors was influenced by the structure of the vector in transfected cells. No differences in transgene expression or DNA molecular structures were observed when AAV ITRs were included to flank the hAAT expression cassette in both ccDNA- and LDNA-treated animals. Linear DNA transfection provides an approach for achieving long-term expression of a transgene in vivo.

Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer.

Sustainable correction of severe human genetic disorders of self-renewing tissues, such as the blistering skin disease junctional epidermolysis bullosa (JEB), is facilitated by stable genomic integration of therapeutic genes into somatic tissue stem cells. While integrating viral vectors can achieve this, they suffer from logistical and biosafety concerns. To circumvent these limitations, we used the Sleeping Beauty transposable element to integrate the LAMB3 cDNA into genomes of epidermal holoclones from six unrelated JEB patients. These cells regenerate human JEB skin that is normalized at the level of laminin 5 protein expression, hemidesmosome formation and blistering. Transposon-mediated gene delivery therefore affords an opportunity for stable gene delivery in JEB and other human diseases.

Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo.

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10(11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than approximately 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.

Human cell receptor CD46 is down regulated through recognition of a membrane-proximal region of the cytoplasmic domain in persistent measles virus infection.

Monkey cells persistently infected by measles virus (MV) Biken strain (Biken-CV-1 cells) showed no cytopathic effects and lacked surface expression of a homolog of human cell receptor, membrane cofactor protein CD46. Transfection of a human CD46 gene into these cells induced extensive cell fusion, indicating that down regulation of the endogenous CD46 homolog was essential for the maintenance of a noncytopathic mode of infection. Surface expression of the exogenously introduced human CD46 was also drastically down regulated in the persistently infected cells compared with uninfected cells. The down regulation was specific for CD46 and did not affect surface expression of exogenously introduced CD4. Exogenous human CD46 was synthesized efficiently in the persistently infected cells, but it did not accumulate on the cell surface. Fusion of Biken-CV-1 cells required the extracellular hemagglutinin (H-protein)-binding domain but not the cytoplasmic domain. Replacing the transmembrane and cytoplasmic domains of CD46 with a glycosylphosphatidylinositol anchor did not prevent cell fusion but completely alleviated down regulation of the glycosylphosphatidylinositol-anchored CD46 in Biken-CV-1 cells. Deletion analyses revealed that the membrane-distal sequences of the CD46 cytoplasmic domain were not only unnecessary but also inhibitory for CD46 down regulation. By contrast, the six amino acid residues proximal to the membrane contained a sequence required for CD46 down regulation in the persistently infected cells. These results indicate that CD46 is down regulated in the persistently infected cells by a mechanism that recognizes a membrane-proximal sequence in the CD46 cytoplasmic domain.

High affinity YY1 binding motifs: identification of two core types (ACAT and CCAT) and distribution of potential binding sites within the human beta globin cluster.

PCR-assisted binding site selection was used to define the sequence characteristics of high affinity YY1 binding sites. Compilation of the sequences of 189 selected oligonucleotides containing high affinity YY1 binding sites revealed two types of core sequence: ACAT and CCAT. ACAT cores were surrounded by other invariant nucleotides, forming the consensus GACATNTT. A search of the 73 kb human beta-like globin cluster with this consensus revealed eight matching motifs, six of which were located within 1-3 kb upstream of the gamma and beta genes. CCAT-type cores were more variable in surrounding sequence context; the consensus VDCCATNWY was found to fit 89% of the selected CCAT-containing oligonucleotides. A search of the human beta globin cluster with CCAT consensus sequences revealed 171 potential YY1 binding sites. Several of these were tested directly in gel shift assays and confirmed as high affinity YY1 binding sites. Finally, a strategy called motif-based phylogenetic analysis was employed to determine which of the 179 total sites are evolutionarily conserved. This analysis permits the detection of functionally conserved binding sites despite sequence differences present between the two species. The 21 conserved sites identified will serve as important starting points in further dissection of the possible role of YY1 in globin gene regulation.